Uric Acid (Liquid) Reagent Set - BrandSD

Uric Acid (Liquid)
Reagent Set
Intended Use
For the quantitative determination of Uric Acid in serum. For in vitro
diagnostic use only.
Clinical Significance
The determination of uric acid in serum is most commonly performed for the
diagnosis of gout. Increased uric acid levels are also found in leukemia,
polycythemia, familial idiopathic hyperuricemia, and conditions associated
with decreased renal function.
Test Summary
Uric Acid has been determined by phosphotungstate methods, 1 variations of
the phosphotungstate method 2 and iron reduction methods. 3,4 The above
methodologies are influenced by many substances in their procedures as
well as many contaminating substances on glassware, etc. 5 The enzyme
Uricase has been widely used for Uric Acid determinations because of its
improved specificity. 6,7 Recently, hydrogen peroxide, a by-product of the
Uricase-Uric Acid reaction, has been coupled to other enzymatic reactions to
yield a colorimetric end product. The present procedure uses the coupling of
4-aminoantipyrine (4-AAP), 2-Hydroxy-3,5-Dichloro-benzenesulfonate
(HDCBS), and hydrogen peroxide in the presence of peroxidase to yield a
chromagen measured at 520nm.
Principle
Uricase
Uric Acid + O2 + 2H2O --------------------------‣ Allantoin + CO2 + H2O2
POD
2H2O2 + 4-AAP + HDCBS ------------------‣ Chromagen + 4H2O
Uric Acid is oxidized by Uricase to allantoin and hydrogen peroxide. HDCBS
+ 4-AAP + hydrogen peroxide, in the presence of peroxidase, produces a red
chromagen that is measured at 520nm. The absorbance at 520nm is
proportional to the concentration of Uric Acid in the sample.
Reagent Composition
Uric Acid reagent: 4-AAP >0.2mM, HDCBS 2mM, Uricase (Microbial) >150
U/L, Peroxidase (horseradish) >2,500 U/L, Buffer, pH 8.1 ± 0.1, Non-reactive
stabilizers.
Reagent Preparation
The reagent is ready to use.
Reagent Storage and Stability
The reagent set is stored at 2-8°C. Under proper storage the reagent will
remain stable until the indicated expiration date.
Precautions
1. This reagent set is for in vitro diagnostic use only.
2. The reagent should not be used if: The reagent is turbid or contains
obvious microbial growth. The reagent blank has an absorbance of
0.500 or greater at 520nm. A pink color is normal for this reagent.
3. All specimens and controls should be handled as potentially infectious,
using safe laboratory procedures. (NCCLS M29-T2) 8
Specimen Collection and Storage
1. Unhemolyzed serum is recommended.
2.
Uric Acid in serum is stable for three days at 2-8°C and up to six months
when frozen. 9
3. Collect specimens per NCCLS document H4-A3. 10
Interferences
1. Elevated ascorbic acid levels can result in falsely depressed Uric Acid
values.
2. Lipemic samples may cause falsely elevated Uric Acid levels.
3. Hemoglobin to 100 mg/dl has been demonstrated to have a negligible effect
(

Uric Acid (Liquid)
Reagent Set
3. Lipemic samples will give falsely elevated results and a serum blank
must be run. Serum Blank: Add 0.025ml (25ul) of sample to 1.0ml
water. Zero spectrophotometer with water. Read and record
absorbance and subtract reading from test absorbance. Calculate as
usual.
Calibration
Use an NIST-traceable Uric Acid standard (5.0 mg/dl) or serum calibrator.
The procedure should be calibrated according to the instrument
manufacturer’s calibration instructions. If control results are found to be out
of range, the procedure should be re-calibrated.
Calculations
A = Absorbance
A (Unk) x Conc. of Std (mg/dl). = Uric Acid (mg/dl)
A (Std)
Example: A (Unk) =0.126, A (Std) = 0.100, Conc. of Std = 5 mg/dl.
Then: 0.126 x 5 = 6.3 mg/dl
0.100
SI Units (mM/L)
To convert to mM/L, multiply the result (mg/dl) by 10 to convert dl to L and
divide by 168 (the molecular weight of Uric Acid).
Mg/dl x 10 = mM/L
168
Example: 6.3mg/dl x .0595 = 0.374mM/L
mg/dl x .0595 = mM/L
Quality Control
Serum controls with known normal and abnormal uric acid values should be
run routinely to monitor the validity of the reaction. These controls should be
run at least with every working shift in which uric acid determinations are
performed. It is strongly recommended that each laboratory establish their
own frequency of control determination.
Expected Values
2.5 - 7.7mg/dl 9
It is strongly recommended that each laboratory establish its own normal
range.
Performance
1. Assay Range: 0 - 20 mg/dl
2. Comparison: Results obtained with this reagent (y), in 132 samples
ranging in Uric Acid from 1.9-10.5 mg/dl, were compared with those
obtained in the same samples using a dry-powder reagent (x) based on
the same methodology. The correlation coefficient was 0.999 and the
regression equation was y=1.00x-0.02. (Sy.x=19.66).
3. Precision: Precision studies were performed following the guidelines
contained in NCCLS document EP5-T2. 12
Within Day (n=20)
Day to Day (n=20)
Mean S.D. C.V.% Mean S.D. C.V.%
6.0 0.06 1.0 6.1 0.16 2.63
6.9 0.04 0.58 7.2 0.16 2.24
9.9 0.06 0.60 10.2 0.31 3.05
4. Sensitivity: The sensitivity of this reagent was investigated by reading the
change in absorbance at 520nm for a saline sample, and two serum samples
with known concentrations. Ten replicates of each sample were performed.
The results of this investigation indicated that, on the analyzer used, the Uric
Acid (Liquid) reagent showed little or no reagent drift on a zero sample.
Also, that an absorbance change of 0.015 was approximately equivalent to 1
mg/dl of Uric Acid.
References
1. Folin, D., Dennis, W., J. Biol. Chem. 13:469 (1913).
2. Caraway, W.T., Clin. Chem. 4:239 (1963).
3. Morin, L.G., J. Clin. Path. 60:691 (1973).
4. Morin, L.G., Clin. Chem. 20:51 (1974).
5. Brochner-Mortenson, K., Medicine 19:161 (1940).
6. Klackar, H.M., J. Biol. Chem. 167:429 (1947).
7. Praetorius, E., Poulson, H., Scand. J. Clin. Invest 5:273 (1953).
8. NCCLS document “Protection of Laboratory Workers form Infectious Disease
Transmitted by Blood, Body Fluids, and Tissue”, 2 nd Ed. (1991).
9. Henry, R.J., Clinical Chemistry: Principles and Technics, 2 nd Ed.,
Hagerstown (MD), Harper & Row, pp. 531 & 541 (1974).
10. NCCLS document “Procedures for the Collection of Diagnostic Blood
Specimens by Skin Puncture”, 3 rd Ed. (1991).
11. Young, D.S., et al. Clin. Chem. 21:1D (1975).
12. NCCLS document “Evaluation of Precision Performance of Clinical
Chemistry Devices”, 2 nd Ed. (1992).
Manufactured by Pointe Scientific, Inc.
5449 Research Drive, Canton, MI 48188
European Authorized Representative:
Obelis s.a.
Boulevard Général Wahis 53
1030 Brussels, BELGIUM
Tel: (32)2.732.59.54 Fax: (32)2.732.60.03 email: mail@obelis.net
Rev. 12/09 P803-U7581-01